______________________________________                                    Specific Scheme I-A Examples:                                             Reagent Composition                                                       Auxiliary Reagents                                                        Analyte Catalyst A                                                                          Catalyst B Indicator                                                                         Titrant                              ______________________________________                                    cholesterol                                                                       cholesterol                                                                         peroxidase red-ox dye                                                                        Reductant                                    oxidase                                                           cholesterol                                                                       cholesterol                                                                         peroxidase ABTS*   Reductant                                    oxidase                                                           cholesterol                                                                       cholesterol                                                                         peroxidase o-dianis-                                                                         Reductant                                    oxidase              idine                                        cholesterol                                                                       cholesterol                                                                         peroxidase p-dipheny-                                                                        Reductant                                    oxidase              lamine                                                                    sulfonic                                                                  acid                                         cholesterol                                                                       cholesterol                                                                         peroxidase red-ox dye                                                                        Ascorbic                                     oxidase                      acid                                 cholesterol                                                                       cholesterol                                                                         peroxidase ABTS*   Ascorbic                                     oxidase                      acid                                 cholesterol                                                                       cholesterol                                                                         peroxidase ABTS*   Gentisic                                     oxidase                      acid                                 Uric acid                                                                         Uricase   peroxidase red-ox dye                                                                        Reductant                            Uric acid                                                                         Uricase              ABTS*   Reductant                            Uric acid                                                                         Uricase   peroxidase o-dianis-                                                                         Reductant                                                         idine                                        Uric acid                                                                         Uricase   peroxidase p-diphenyl-                                                                       Reductant                                                         lamine                                                                    sulfonic                                                                  acid                                         Uric acid                                                                         Uricase   peroxidase red-ox dye                                                                        Ascorbic                                                                  acid                                 Uric acid                                                                         Uricase   peroxidase ABTS*   Ascorbic                                                                  acid                                 Uric acid                                                                         Uricase   peroxidase ABTS*   Gentistic                                                                 acid                                 ______________________________________                                     *ABTS is 2,2'-azino-di-(3-ethyl-benzothiazoline-6-sulphonic acid.         NOTE: In any of the above, the red-ox dye may be any compound oxidizable  by H.sub.2 O.sub.2 (plus peroxidide) which is reconverted to reduced form by titrant (reductant) and is visibly colored in either, but not both, of its reduced and oxidized forms.

SCHEME I-B

1. Analyte + reagent + reduced indicator→Oxidized Indicator

2. Oxidized indicator + titrant→reduced indicator

______________________________________                                    Specific I-B Examples:                                                    Analyte ****                                                                        Reagent       Indicator Titrant                                 ______________________________________                                    Hemoglobin                                                                          Organic       Ox-red    reductant ***                                     peroxide *    indicator **                                      Peroxidase ****                                                                       "             "         "                                     Blood ****                                                                            "             "         "                                     Glutamic- alanine         "         "                                     pyruvic   alpha-ketoglutarate                                             transaminase                                                                        lacticdehydrogenase                                                       reduced                                                                   nicotinamideadenine                                                       dinuclectide                                                              (NADH)                                                          Glutamic- aspartic acid   "         "                                     oxaloacetic                                                                         alpha-ketoglutaric                                              transaminase                                                                        acid                                                                      malic acid                                                                dehydrogenase                                                             (NADH)                                                          * organic peroxides:                                                      Cumene hydroperoxidez****                                                 ** Indicators:                                                            o-dianisidine                                                             p-toluidine                                                               ABTS (supra)                                                              p-diphenylamine sulfonic acid                                             o-tolidine                                                                neutral red (cert.)                                                       janus green B (cert.)                                                     2,6-dibromoindophenol sodium salt (pract.)                                N,N-dimethylindoaniline (pract.)                                          **** See U.S. Pat. Nos. 3,012,976; 3,092,463 3,252,762; and                3,290,117. -                                                                   Indophenol sodium salt (pract.)                                     2,3',6-trichloroindophenol sodium salt (pract.)phenosafranine                                                            5,5'-indigodisulfonic acid disodium salt (pract.)                         nile blue A (cert.)                                                       methylene blue (cert.)                                                    thionin (cert.)                                                           brilliant cresyl blue (cert.)                                             2,6-dichloroindophenol sodium salt                                        N-(p-methoxyphenyl)-p-phenylene-diamine                                   hydrochloride                                                             3,3'-dimethoxybenzidine (pract.)                                          3,3'-dimethoxybenzidine dihydrochloride                                   N,N-dimethyl-p-phenylenediamine (pract.)                                  N,N'-diphenylbenzidine (pract.)                                           diphenylamine                                                             4-(p-ethoxyphenylazo)-m-phenylenediamine                                  monohydrochloride                                                         p-diphenylaminesulfonic acid sodium salt                                  barium diphenylaminesulfonate                                             o-tolidine                                                                xylene cyanole FF (techn.)                                                N-phenylanthranilic acid                                                  tris (1,10-phenanthroline)iron(II) sulfate                                solution                                                                  dicyanobis (2,2'-bipyridine) iron                                         dicyanobis (1,10-phenanthroline) iron                                     benzopurpurin 4B                                                          naphthol blue black                                                       *** Titrants:                                                             Gentisic acid                                                             Ascorbic acid                                                             Hydroquinone                                                              Pyrogallol                                                                Hydroxylamine                                                             sodium nitrite                                                            sodium bisulfite                                                          sodium thiosulfate                                                        cysteine                                                                  hydrazine                                                                 ferrous ion, and complexes thereof                                        cuprous ion, and complexes thereof

______________________________________                                    Examples                                                                   Analyte  Auxiliary Reagents                                                                        Indicator Titrant                               ______________________________________                                    Lactic acid                                                                         lactic acid     Ox-red    Oxidant                                         dehydrogenase   indicator (list) **                                       NAD (nicotinamide                                                                         (list) *                                                  adenine dinucleotide)                                           Lactic acid                                                                         lactic acid       "         "                                   dehydrogenase                                                                       NAD                                                             Triglycerides                                                                       lipase            "         "                                             glycerol dehydrogenase                                                    NAD                                                             Hydroxybutyric                                                                      hydroxybutyric acid                                                                         "         "                                   acid      NAD                                                             dehydrogenase                                                             * Indicator List:                                                         same as I-B above.                                                        ** Oxidant List:                                                          1. cumene hydroperoxide + horse radish peroxidase                         (auxiliary reagent)

SCHEME III

Compositions that effect a titration of the analyte against specific complexigenic compounds or substances.

SCHEME III-A

Compositions in which titrant forms an unreactive complex with analyte, thus preventing reaction of analyte with auxiliary reagents unless analyte concentration exceeds complexigenic compound concentration.

______________________________________                                    Examples:                                                                  Analyte   Titrant        Auxiliary Reagents                              ______________________________________                                    Ketones **                                                                         sodium bisulfite sodium nitroprusside                                                      glycine                                         Proteins **                                                                        sodium salycilate                                                                          tetrabromophenol                                  "      sodium dodecyl sulfonate                                                                     "                                             Albumin  sodium salycilate                                                                          brom cresol green                                 "      sodium dodecyl sulfonate                                                                     "                                               "      sodium salycilate                                                                          o((p-hydroxyphenyl)                                                       azo)-benzoic acid                                 "      sodium dodecyl sulfonate                                                                     "                                             Bilirubin                                                                          simple aldehydes dichloroaniline                                                           diazonium salt                                    "      benzaldehyde       "                                             Urobilinogen ***                                                                   simple aldehydes p-dimethylamino-                                                          benzaldehyde                                      "      benzaldehyde       "                                             ______________________________________                                     * See U.S. Pat. No. 3,212,855                                             ** See U.S. Pat. Nos. 3,095,277 and 3,438,737.                            *** See U.S. Pat. Nos. one or more of following: 3,050,373; 3,123,443;    3,122,420; 3,453,180; 3,585,001; 3,585,004; and 3,447,905.

SCHEME III-B

Compositions in which analyte reacts with a specific binding substance (titrant) and renders at least a portion of said substance undetectable by a subsequent indicating auxiliary reagent.

GENERAL SCHEME: (III-B)

Reagent zones each contain a different amount of titrant. Upon exposure to biological fluid, containing analyte, one or more zones will be completely reacted with analyte, and will be unable to react with subsequent indicator reagent. For fluids containing low concentrations of analyte, many or all zones will remain detectable. For fluids containing large quantities of analyte, most or all zones will be rendered non-detectable.

______________________________________                                    Specific Examples of III-B:                                               Analyte  Titrant      Indicator Reagent(s)                                ______________________________________                                    1.  Antigen  antibody     labeled antigen                                       "        "          radioactive antigen                                   "        "          enzyme-labeled antigen                                                    plus appropriate                                                          substrates to generate                                                    acolored product                               2.    "        "          horseradish peroxidase-                                                   labeled antigen, cumene                                                   hydroperoxide, ABTS (supra)                     3.    "        "          horseradish peroxidase-                                                   labeled antigen, sodium                                                   perborate, ABTS (supra)                         4.  Ligand   specific-ligand-                                                                       same as 1 above, but                                         binding protein                                                                        substitute "ligand" for                                                   "antigen"                                       5.    "        "          same as 2 above,                                                          substituting again                              6.    "        "          same as 3 above,                                                          substituting                                    ______________________________________                                     NOTE: Relative to III-B example, enzyme labels in such assays are well    known; see for example U.S. Patent Nos. 3,791,932; 3,839,153; and         3,850,752. In these patents the claims all relate to methods dependent    upon measuring the amount of enzyme label found after certain separation  procedures. In my invention, I do not measure amounts of enzyme label, bu only use it as a gross indicator of whether or not all antibody combining sites have been reacted.

The following examples describe the manner and process of making and using this invention and set forth the best mode contemplated by the inventor of carrying out the invention but are not to be construed as limiting. All parts are by weight unless otherwise indicated.

EXAMPLE I

A. To an appropriate vessel there is charged 300 microliters of a solution of cholesterol oxidase (1.0 IU/ml, in phosphate buffer 0.1M, pH 7), 300 microliters of solution of cholesterol esterase (1.0 IU/ml in phosphate buffer, pH 7), 150 microliters of a solution of horseradish peroxidase (30,000 IU/ml in 0.01M phosphate buffer, pH 7), 300 microliters of 2,2'-azino-di-(3-ethyl-benzothiazoline-6-sulphonic acid) in solution [prepared by dissolving 50 milligrams of 2,2'-azino-di-(3-ethyl-benzothiazoline-6-sulphonic acid) (Boehringer Manneheim Corporation, New York, New York, product No. 15594) in 1.0 milliliters of 0.1M phosphate buffer (pH 7)] and 0.1M phosphate buffer (pH 7) quantum sufficient to make a total of 1,250 microliters. The mixture is thoroughly admixed to obtain a cholesterol indicating reagent solution.

B. In an appropriate vessel 0.5 grams of 2,5-dihydroxybenzoic acid is slurried in 4 milliliters of water. To the slurry there is then added with stirring a solution of 10% sodium hydroxide in water until a clear solution is obtained. This is used as a stock 10% solution. A working 1% solution is prepared by appropriate dilution with 0.1M phosphate buffer (pH 7).

C. Four separate appropriate vessels are each charged with 80 microliters of the indicating reagent solution prepared in (A) above. In each vessel there is then added with mixing, various proportions of the 1% 2,5-dihydroxybenzoic acid solution prepared in (B), supra, and varying amounts of buffer as described in (A) above to obtain cholesterol indicating reagent solutions, each of which will indicate visually when exposed to various minimum concentrations of cholesterol in solution, by a color change of from substantially colorless to colored. The four vessels are identified by the letters A through D inclusive. The concentration of 2,5-dihydroxybenzoic acid and buffer in each vessel and the minimum cholesterol concentration of a solution which each reagent composition A through D inclusive will indicate by a color change are shown in Table I below.

              TABLE I                                                     ______________________________________                                                                     Percent of                                                                Minimum                                                                   Cholesterol                                                      Concen-  Concentration inBuffer   2,5-DHBA  tration  Solution Required                        Ves- (Micro-  (Micro-   of       for Indication                           sel  liters)  liters)   2,5-DHBA to Occur                                 ______________________________________                                    A    12        8         80 mg/dl                                                                          200 mg/dl                                B    8        12        120 mg/dl                                                                          300 mg/dl                                C    4        16        160 mg/dl                                                                          400 mg/dl                                D    0        20        200 mg/dl                                                                          500 mg/dl                                ______________________________________

EXAMPLE II

A series of five sets of circular discs are prepared, each set consisting of four discs and each disc being fashioned from #52 Whatman filter paper by cutting out a circle, 4 mm, in diameter. Each disc within each set is then wetted with about 0.002 ml of a different one of the reagent solutions A, B, C and D, respectively, prepared in Example I, supra. The wet discs are allowed to air-dry. Each disc is calculated to contain, on drying, approximately 0.00038 IU of cholesterol oxidase, 0.00038 IU cholesterol esterase, 5.6 IU of peroxidase, 19.2 micrograms of 2,2'-azine-di-(3-ethyl-benzothiazoline-6-sulphonic acid) and varying proportions of 2,5-dihydroxybenzoic acid. The dry discs of each set are then mounted on a single paper strip, and each disc labeled a, b, c or d to correspond to the particular reagent A, B, C and D, respectively, employed in its preparation. Four of the paper strips each bearing a separate set of the four discs a-d is then immersed in one of four aqueous solutions containing 200, 300, 400, and 500 mg/dl cholesterol respectively. The fifth strip is a control strip and is immersed in distilled water. Each strip is immersed in the solution for a period of about one second and then withdrawn. Each strip is observed for a change of color in the discs mounted thereon as an indication of the presence or absence of cholesterol. The expected results are given in Table II below, with the proportion of 2,5-dihydroxybenzoic acid calculated to be present in each disc.

              TABLE II                                                    ______________________________________                                                               Proportion of                                        % ofCholesterol     2,5-dihydroxy-                                 Strip in Immersion         benzoic Acid                                                                         Color                               No.   Solution      Disc   (mcg) per disc                                                                       Change                              ______________________________________                                    1      200 mg/dl                                                                               a       1.6       Yes                                                     b       2.4       No                                                      c       3.2       No                                                      d       4.0No                                 2      300 mg/dl                                                                               a       1.6       Yes                                                     b       2.4       Yes                                                     c       3.2       No                                                      d       4.0No                                 3      400 mg/dl                                                                               a       1.6       Yes                                                     b       2.4       Yes                                                     c       3.2       Yes                                                     d       4.0No                                 4      500 mg/dl                                                                               a       1.6       Yes                                                     b       2.4       Yes                                                     c       3.2       Yes                                                     d       4.0       Yes                                5 (Control)                                                                         0.0%                                                                                 a       1.6       No                                                      b       2.4       No                                                      c       3.2       No                                                      d       4.0       No                                 ______________________________________

glutamic-oxaloacetic transaminase

glutamic-pyruvic transaminase

lactic acid dehydrogenase

hydroxybutyric acid dehydrogenase

creatine phosphokinase

their determinations in a biological fluid may be determined.

The indicator of the invention and the method of the invention will now be further illustrated by reference to the specific embodiments shown in the accompanying drawings.

In FIG. 1 there appears a perspective view of anindicator 20 of the invention. Theindicator 20 comprises a support member which is shown in FIG. 1 as aflat strip 18 and which preferably is bibulous material such as an absorbent paper strip. Disposed on thestrip 18 by impregnation thereon in a manner so as to form the

numerals

1, 2, 3, and 4 are

reagents

11, 12, 13 and 14 respectively. Reagent 11 is a composition such as composition A of Example I, supra. and will indicate by color change when a minimum cholesterol concentration of 0.2% is present in a solution brought into contact with the reagent.

Reagents

12, 13 and 14 correspond to the reagent compositions B, C and D respectively of Example I, supra. and will indicate the minimum cholesterol concentrations of 0.3%, 0.4% and 0.5% in cholesterol containing solutions, respectively. The disposition of the reagent compositions on thesupport member 18 may of course be in any convenient indicia. For example, the minimum concentration indicated by a given reagent may be the form of disposition, i.e.; for example, reagent 11 above described may be disposed in the configuration "0.2%". The

numerals

1, 2, 3 and 4 selected for illustration of FIG. 1 is especially useful when the indicator device is to be employed for determining the chemical concentration in broad ranges. Such indicia conform to the to the practice of referring to a 0.2% concentration as a "plus 1" level, a 0.3% concentration as "plus 2", a 0.4% concentration is "plus 3" and a 0.5% concentration as "plus 4".

Prior to use, the

symbols

1, 2, 3 and 4 employed in the embodiment of FIG. 1 may be invisible to view or only slightly decipherable through a slight discoloration uponstrip 18. The broken lines indicating the

numerals

3 and 4 are intended to show that the

numerals

3 and 4 are not clearly decipherable as originally formed onmember 18. Upon exposure however to a chemical solution by immersion therein, the reagents react chemically to produce a color. If the chemical concentration in the tested solution exceeds the minimum concentration for which the reagent will give a color indication, such color change occurs. In the FIG. 1numerals 1 and 2 are shown in distinct dark tones and illustrate the indication obtained following immersion ofindicator 20 in a solution of a chemical containing a chemical concentration of from between 0.3% and 0.4%.Reagents 11 and 12 have reacted to indicate a "plus 2" concentration, i.e.; the highest number developed. The development ofreagents 11 and 12 to produce a clear and distinct color change forming distinct solid numerals gives a direct reading to the user of the minimum chemical concentration in the solution tested. Thus, the method of the invention is carried out by immersing that portion ofindicator 20 which bears

reagents

11, 12, 13 and 14, into a chemical containing solution momentarily, withdrawing it and visually observing the indicia registered. It will be observed that the operator does not have to carry out a comparison of colors registered, with standard charts to determine the concentration of the tested solution, as is necessary with prior art disposable devices. When the preferred composition of the invention is employed as the

reagents

11, 12, 13 and 14, clear and distinct indications of concentration are obtained within about one minute. The indicia registered on the indicator are relatively stable and will last for a relatively long time so that at an interval of up to at least 72 hours after immersion and withdrawal ofindicator 20, the user may refer back to recheck the test results. This is not possible with prior art devices.

The embodiment illustrated in FIG. 1 may obviously be modified in many respects to meet specific needs. The degree of sensitivity of each reagent may be modified to meet specific needs. Different ranges and sensitivities of the indicator devices may be obtained by selection of the reagent sensitivities and the number of different reagent compositions applied to the support member. The support member,strip 18 may also be dyed a particular color to either blend with the reagent compositions prior to their indicating the presence of chemicals or they may be dyed a color to provide a very sharp contrast when the reagent reacts to produce a registration.

As further examples of modifications which may be made to the basic indicator shown in FIG. 1, reference is now made to FIG. 2 which shows an isometric view ofindicator 22 which may also comprise asupport member 18 as described above and upon which there have been mounted "plus-shaped" blocks 5 of a bibulous material. Eachblock 5 has been saturated with a solution of a

reagent

11, 12, 13 and 14 respectively as described above in conjunction with FIG. 1 and then dried before fixation uponsupport member 18. Whenindicator 22 is immersed briefly in a chemical containing solution the reagents carried onblocks 5 react as described above, and if the chemical level exceeds the minimum indicating sensitivity of the particular reagent, a color change in theblock 5 carrying that reagent is visually observable. By counting the "pluses" which have changed color, the operator can read the device directly. In thedevice 22, as shown, a "plus 2" has been indicated, i.e.; the device was exposed to a solution containing between 0.3% and 0.4% of, for example, cholesterol.

FIG. 3 is an alternate embodiment similar todevice 22 of FIG. 2, differing essentially in that the shape of thebibulous block 6 is a rectangle rather than a cross. This embodiment is operated in the same way as the device of FIG. 2 and as shown here has been exposed to a solution of a chemical in the "plus 3" concentration range, i.e.; the solution tested had a chemical concentration of between 0.4% to 0.5%.

The embodiment of FIGS. 2 and 3 are particularly advantageous when thesupport member 18 is a hydrophobic material such as, for example, polyethylene. The

blocks

5 and 6 will each then absorb a predetermined volume of the biological fluid being tested, upon immersion. When the

indicator reagents

11, 12, 13 and 14 have been deposited on

blocks

5 and 6 by saturation of said

blocks

5 and 6 with the

indicator reagents

11, 12, 13 and 14 in solution followed by evaporation of the reagent solvent, the volume of test solution absorbed in

blocks

5 and 6 on immersion in test solution will be substantially equal to the volume of solvent evaporated from the

blocks

5 and 6 to deposit the indicator reagent. This is the preferred ratio of volume of reagent in solution to volume of test solution absorbed as previously described, and results in a highly accurate, precise indicator device of the invention.

FIGS. 4a, 4b, 4c and 4d illustrate still another embodiment of the invention. The indicator device is referred to generally by the numeral 24 in these Figures. In FIG. 4a,indicator 24 is shown to comprise asupport member 18 as described previously, but bearing

reagents

11, 12, 13 and 14 as previously described in a sequence so that reagent 11 is disposed in the form of a symbol 1,reagent 12 is disposed in the form of asymbol 2 and is also disposed in a zone surrounding the symbol 1 formed by a reagent 11. In asimilar manner reagent 13 is disposed in the form of asymbol 3 and also in the zone surrounding thesymbol 2.Reagent 14 is disposed in the form of asymbol 4 and also in the zone surrounding thesymbol 3. In this embodiment, exposure to a chemical containing solution of a specific level will indicate numerically the minimum concentration of chemical in the tested solution and will obliterate numeral symbols for any lower concentrations of chemical. For example, FIG. 4a shows by broken lines the zones and disposition into indicia of the

reagent compositions

12, 13 and 14 not registering the specific concentrations of chemical to which they are sensitive. The numeral 1 appears in bold outline as indicative of a "1" concentration of chemical, meaning theindicator 24 as shown in FIG. 4a has been exposed to a solution containing at least 0.2% of chemical.

Referring to FIG. 4b, there is seen the registration obtained by immersion, briefly, ofindicator device 24 in a test solution containing between 0.3% and 0.4% chemical concentration. The registration of thenumeral 2 and obliteration of the also developed numeral 1. This is a clear indication to the user of a "plus 2" concentration in the tested solution. FIG. 4c shows the indicia registered whenindicator 24 was immersed in a solution containing between 0.4% and 0.5% chemical and FIG. 4d shows the indicia registered upon immersion ofindicator 24 in a solution containing more than 0.5% chemical, i.e.; a "plus 4" reading.

FIG. 5 is an isometric view of a preferred indicator of the invention shown after use by immersion in a solution containing between 0.3% and 0.4% chemical. Theindicator 26 comprises as a support member a bibulous material 30 which has been laminated to astrip 32 of stiff supporting material such as polymeric plastic for example a strip of polyethylene, polypropylene or like polymeric material.

Apertures

35, 36, 37 and 38 have been cut into supportingstrip 32 in the form of positive signals in those areas overlying support member 30 which have been impregnated with the

various reagents

11, 12, 13 and 14 previously described. In this manner, the various zones of support member 30

bearing reagents

11, 12, 13 and 14 are viewed through the corresponding

apertures

35, 36, 37 and 38 respectively. In addition, an area or zone of support member 30 has also been impregnated with areagent 9 which is sensitive to an acid pH. Thereagent 9 impregnated zone of support member 30 is visible throughaperture 39 cut intostrip 32. The purpose of the zone impregnated withreagent 9 is to serve as a control. When theindicator 26 is immersed in acid biological fluids, contact of the fluid withreagent 9 registers a color change. The user is then assured that all of the reagent zones belowreagent 9 on member 30 have made contact with the solution, and therefore reagents 11, 12, 13 and 14 have been exposed to the fluid specimen.

FIG. 6 is a cross-sectional view alonglines 6--6 of FIG. 5 and shows in greater detail the mounting of support member 30 beneath the

cutaway zones

35, 36, 37, 38 and 39 in rigid supportingstrip 32. This embodiment, as illustrated in FIGS. 5 and 6, immediately provides the user, upon immersion in a fluid specimen, with a visual indication of chemical level registered in terms of plus units which are readily calculated by counting the darkened indicia. Thus the illustrated device of FIGS. 5 and 6 has registered a "plus 3" value of chemical concentration, in the illustration.

FIG. 7 is a view in perspective of another indicator embodiment of the invention which is particularly useful for precise determinations of the concentration of a substance dissolved in liquids, which potentially may have a broad rang of possible concentrations. For example, the indicator shown generally by the numeral 70 is useful in measuring the cholesterol concentration of mammalian blood specimens. Indicator 70 comprises a transparent or highly translucent support member 72 such as a sheet of transparent cellulose acetate, a film of polyethylene, transparentized paper or like materials. Disposed on member 72 are a variety of reagents, such as

reagents

11, 12, 13 and 14 previously described, with reagent compositions of intermediate sensitivities disposed between them as illustrated in the figure. Prior to exposure to a solution of cholesterol, for example blood serum, the zones covered by the various reagent compositions are relatively colorless. A sample of blood serum is wiped over the surface of the reagent bearing member 72, causing those reagents which indicate at the level of cholesterol concentration found in the blood specimen to undergo a visible color change. The indicator 70 is "read" by a hand held optical aid or by inserting it in a light projector with a reticule 75. Reticule 75 projects a reference grid, which is superimposed by projection over the indicator 70, whereby the indication registered on 70 is readily observed in a magnified and projected image. Of course when the indicator is to be read directly without the aid of a light projector, there is no need for a transparent support member 72.

The embodiment of the invention described above in relation to FIG. 7 is representative of another facet of the invention which comprises the use of reading aids, such as optical and electromechanical devices to assist in reading the indicators of the invention. More specifically the indicators of the invention function basically by the development of a color change, darkening or opacifying of a zone upon the support member component of the indicator device. Reading of the device is dependent upon an observation of a particular quantity of reacted or indicated zones. The actual "reading" of the indicators can therefore be performed mechanically by instruments designed to count symbols through the use of lenses, mirrors, filters, projectors, detectors and the like which can discriminate by means of light transmission, light reflectance, light scattering, electrical conductivity, radioactivity, etc. Thus, the reading of the indicator devices of the inventor may be carried out mechanically with the aid of photo scanners and like devices. This facet of the invention is particularly advantageous for use of the indicator devices of the invention bearing a large number of different reagent zones, each zone being of a microdot dimension or some dimension not readily observable with the naked eye. Such devices are particularly advantageous in that they use a minimum of reagent material, are fairly compact and can provide readings of great accuracy over wide ranges of possible substance concentrations in the solutions being tested.

The indicators of the invention may be used in ways not limited to those described in the foregoing. For example, before or after exposure to the test specimen, they may be exposed to reagents not previously incorporated in the reactive zones, or to a drying process, e.g. application of heat, or other chemical or physical agents selected for the purpose of developing or enhancing the appearance of visual or otherwise detectable indications.